Alpha-1 antitrypsin (AAT) deficiency, due to the "PiZZ" mutation, results in life-threatening lung and liver diseases in children and adults. Fortunately, the lung disease can be prevented by gene addition therapy. However, this strategy could not halt liver disease progression due to the accumulation of mutant PiZZ protein in the endoplasmic reticulum of liver cells rather than normal secretion into the blood and body fluids. Therefore, gene therapy for the PiZZ-associated liver diseases should be focused on long-term elimination/correction of mutant protein at the DNA and/or RNA levels. Small interference RNA (siRNA) represents a promising approach to suppress PiZZ and a persistent DNA vector-based siRNA could prolong this effect for years (Aim 1). Adeno-associated virus 2 (AAV2) vectors have been utilized for treatment of AAT deficiency in pre- and clinical trials. In the liver, transduction by recombinant (r) AAV2 induces sustained gene expression;however, only 5-10% of liver cells are transduced even at a high particle to cell ratio. Recent studies, including ours, demonstrated that other AAV serotypes, mainly AAV6, AAV8 and AAV9 are able to transduce liver cells more efficiently than AAV2. To further enhance this transduction, we have developed a novel AAV vector that packages a double-stranded (ds) genome. These vectors by-pass the rate limiting step of second-strand synthesis resulting in both increased and earlier transgene expression (up to two weeks earlier than the traditional single-stranded (ss) vector). Specifically in liver, we have been able to demonstrate dsAAV 2 transduction as high as 90%, and dsAAV 8 with over 95% transduction using a lower total dose. Even though AAT expression can be detected in blood from AAV transduced muscles, liver is a natural organ to produce AAT, and higher transgene expression has been observed in AAV transduced liver compared to muscle. Therefore, suppression of PiZZ gene expression along with the successful gene addition strategy should eliminate both disease manifestations, liver and lung respectively. To avoid wtAAT mRNA degradation induced by siRNA/PiZZ and to increase overall AAT synthesis, we will create an optimized AAT gene based on the degeneracy of the genetic code (Aim 2). It should be noted that although AAV2 and AAV8 transduce the liver with high efficiency the overall tropism is broad and certainly not restricted to liver cells. Several steps including viral binding, endocytosis, trafficking and uncoating are required for cell specific transduction and we have begun to relate structure to function using a shuffled serotype capsid library. Supported by our preliminary data, it is possible to evolve a lab strain of AAV that may have superior, yet restricted, liver transduction to reduce siRNA "off-target" effect in other tissues. It also suggests that specific capsid domains responsible for efficient liver transduction can be identified (Aim 3). PUBLIC HEALTH RELEVANCE: Adeno-associated virus (AAV) is a promising delivery vector for alpha-antitrypsin (AAT) deficiency (Alpha-1) gene therapy. AAV is able to confer long-term stable expression of a therapeutic gene and does not cause any known disease. Recently, 12 types of AAV have been isolated and AAV2 is the best characterized for its biology and as a gene delivery system. Only 5-10% of liver cells can express the AAT gene following infection with AAV2 vectors. AAT liver expression is much higher following infection with AAV1, 5, 8 &9 vectors. This suggests that different types of AAV use different cellular pathways for infection (i.e. cell surface binding, movement of the virus through the cell, entry of the virus into the nucleus, removal of the viral nucleic acid from its protein shell). AAV is a single-stranded DNA virus. Transcription will ensue from these genomes only after the single-stranded DNA genome is converted to double-stranded (ds) DNA. The use of dsAAV vectors can overcomes this rate limiting step. Recently we have developed the dsAAV vectors which can induce transgene expression much faster and higher than conventional single-stranded AAV vectors. Almost all liver cells are infected with these double-stranded AAV vectors, which is very significant for preventing liver disease development in Alpha-1 patients by using this double-stranded vector to carry therapeutic genes. Since liver disease with AAT deficiency is caused by the existence of mutant AAT inside of liver cells. To destroy this mutant protein, we will deliver siRNA specific for mutant AAT into the liver using dsAAV8 vector (Aim 1). Knocking down of mutant AAT in liver cells only prevent liver disease development and cannot prevent lung damage caused by AAT deficiency. To restore AAT expression, we will infect the liver using dsAAV2 vector to deliver the optimized AAT, which cannot be degraded by mutant AAT/siRNA (Aim 2). Aim 3 describes the development and characterization of new AAV liver-specific variants that will possess superior transduction capacity specifically on liver cells to known serotypes. These new variants will be generated using an approach with in vitro "DNA shuffling" and in vivo selection in liver. This study would be very important to design AAV/AAT vector for future human clinical trials.